Analyzing Obesity and Metformin Effects on Endometrial Proliferation

The effect of obesity and metformin on mouse endometrial proliferation was evaluated in formalin-fixed, paraffin embedded tissue by Ki67 staining (BD Biosciences, San Diego, CA) following the manufacturer’s instructions. The degree of apoptosis in endometrial tissues was determined by IHC staining for cleaved caspase-3 (Asp175; Cell Signaling, Danvers, MA). The sections were counterstained with Mayer’s hematoxylin. The average number of positively-stained Ki67 or Caspase-3 cells in 5–10 high-power microscopic fields were counted per slide, and calculated as: 200×(number of positively stained endometrial cells/total endometrial cells). Tissues were also stained for downstream markers of insulin and IGF1 receptor signaling, including Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), Phospho-p44/42 MAPK (Thr202/Tyr204), and Phospho-Acetyl-CoA Carboxylase (Ser79) (Cell Signaling, Danvers, MA) and scored according to previously published procedures[24 (link)].

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IGLESIAS D.A., ZHANG Q., CELESTINO J., SUN C.C., YATES M.S., SCHMANDT R.E, & LU K.H. (2016). Lean body weight and metformin are insufficient to prevent endometrial hyperplasia in mice harboring inactivating mutations in PTEN. Oncology, 92(2), 109-114.

Publication 2016

Ki67 staining Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), Phospho-p44/42 MAPK (Thr202/Tyr204), Phospho-Acetyl-CoA Carboxylase (Ser79)

Acetyl coa carboxylase Apoptosis Caspase 3 Cells Embedded paraffin Endometrial Formalin Hematoxylin Igf1 receptor Insulin Metformin Microscopic Mouse Obesity P44 mapk Ribosomal protein Tissues

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center

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Variable analysis

independent variables

Obesity
Metformin

dependent variables

Endometrial proliferation measured by Ki67 staining
Apoptosis in endometrial tissues measured by cleaved caspase-3 staining
Downstream markers of insulin and IGF1 receptor signaling, including Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-AKT (Ser473), Phospho-p44/42 MAPK (Thr202/Tyr204), and Phospho-Acetyl-CoA Carboxylase (Ser79)

control variables

Formalin-fixed, paraffin embedded tissue samples
Mayer's hematoxylin counterstaining
Previously published scoring procedures for downstream markers

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